The 174-base bacteriophage phi29 prohead RNA (pRNA) is essential for in vitro packaging of the 19-kilobase pair DNA-gp 3 complex (DNA-g3) into the viral precursor capsid (prohead). pRNA is an integral part of the phi29 DNA packaging motor, one of the strongest molecular motors characterized, pRNA forms a novel cyclic hexamer by intermolecular base pairing of identical molecules. This multimer binds to the head-tail connector of the prohead, the core of the packaging motor, where it appears as a pentameric ring by cryoEM 3-D reconstruction. A multimer of the packaging ATPase gp16 then binds the pRNA to complete the motor. pRNA is hypothesized to function in docking of the DNA-gp3 and the prohead, in recognition of the left end of DNA-gp3 to initiate packaging, and as a component of the DNA translocating ATPase. pRNA exits the DNA-filled head during neck and tail assembly, and it is not a part of the mature virion. Study of the structure and function of this RNA-dependent DNA packaging motor may have general significance for assembly of other viruses, including mammalian viruses. The ultimate goal of the research is to determine the structures and the mechanisms by which pRNA constitutes the phi29 DNA packaging motor to catalyze DNA-gp3 translocation into the prohead. The aims of the current project are to: 1) study the DNA packaging motor comprised of the connector, pRNA and the ATPase gp16 by cryoEM-3D reconstruction and X-ray crystallography; 2) determine the structure of the pRNA intermolecular pseudoknot by NMR; 3) study the packaging motor step size and coordination of pRNA-gp16 motor subunits by the use of laser optical tweezers; 4) determine the role of the N-terminus of gp 10 (connector) in binding pRNA; and 5) identify the DNA packaging RNAs of phages SPP1 and lambda.